The processes involved in tumor growth, progression, and metastasis are mediated by signaling pathways that are activated in cancer cells. The mitogen-activated protein kinase (MAPK) pathway, of which extracellular regulated kinase (ERK) is a downstream member, plays a central role in regulating mammalian cell growth by relaying extracellular signals from ligand-bound cell surface tyrosine kinase receptors such as erbB family, PDGF, FGF, and VEGF receptor tyrosine kinase. Activation of the MAPK/ERK pathway is via a cascade of phosphorylation events that begins with activation of a small guanosine triphosphatase (GTPase), RAS. Activation of RAS leads to the recruitment and activation of RAF, a serine-threonine kinase. Activated RAF then phosphorylates and activates MEK1/2, which in turn phosphorylates and activates ERK1/2. When activated, ERK1/2 phosphorylates several downstream targets involved in a multitude of cellular events including cytoskeletal changes and transcriptional activation.
The MAPK/ERK pathway is one of the most important for cell proliferation and it is believed that this pathway is frequently activated in many tumors. RAS genes, which are upstream of ERK1/2, are mutated in several cancers including colorectal, melanoma, breast and pancreatic tumors. The high RAS activity is accompanied by elevated ERK activity in many human tumors. In addition, mutations of BRAF, a serine-threonine kinase of the RAF family, are associated with increased kinase activity. Mutations in BRAF have been identified in melanomas (60%) (Fecher, L. A., et al., Curr. Opin. Oncol., 2008, 20:183-189), thyroid cancers (approximately 50%) (Robert, R. J., and Der, C. J., Oncogene, 2007, 26: 329103310), and colorectal cancers (Davies, H., et al., Nature, 2002, 417: 949-954; Kohno, M., and Pauyssegur, J., Ann. Med., 2006, 38: 200-211).
KRAS also acts upstream of ERK to regulate proliferation, differentiation, and cell survival. Mutations in codons 12, 13, and 61 of KRAS result in constitutive growth signaling and confers resistance to compounds targeting the EGFR signaling pathway (Andre, T., et al., Annals of Oncology, 2012, 00: 1-8; DeRook, W., et al., Lancit Oncology, 2010, 11: 6753-6762). KRAS mutations are found in 65% of pancreatic cancers, 40% of colon cancers, 20% of lung cancers, and 10% of gastric cancers (Downward, J., Nat. Rev. Cancer, 2003, 3: 11-22; Smalley, K. S. M., Int. J. Cancer, 2003, 204: 527-532). Most patients with KRAS mutations in codons 12, 13, and/or 61 do not respond well to anti-EGFR monoclonal antibody (mAb) therapies (Lièvre, A., et al., Cancer Res., 2006, 66(8):3992-3995).
The mutational status of solid tumors is also becoming increasingly important for identifying the best treatment options for cancer patients (Yokota, T., Anti-Cancer Agents in Medicinal Chemistry, 2012, 12: 163-171). Treatments developed targeting a specific signaling pathway may not be effective when activating mutations are present downstream of the signal transduction pathway. For example, tumors harboring activating mutations in RAS and RAF do not generally respond well to anti-EGFR therapy, in that these mutations are believed to lead to EGFR-independent activation of an intracellular signaling pathway (Messersmith, W. A., and Ahnen, D. J., N. Engl., J. Med., 2008, 359(17): 1834-1836). As such, it is believed that inhibitors targeting proteins further downstream in this pathway may be more effective in treating these cancers.
As such, proteins that lie downstream of RAS and, even further downstream of RAF and MEK, in the MAPK/ERK pathway are potential targets for pharmacological intervention (Downward, J., Nat. Rev. Cancer, 2003, 3: 11-22; Pearson, G., et al., Endocr. Rev., 2001, 22: 153-183; Fecher, L. A., et al., Curr. Opin. Oncol., 2008, 20: 183-189). Gain of function, i.e., activating, mutations in RAS and RAF that lead to constitutive activation of this pathway are frequently observed in human cancers and are associated with high rates of cancer cell proliferation (Id.). It has been reported that activating mutations of RAS were identified in about 25% of all cancers (Smalley, K. S. M., Int. J. Cancer, 2003, 104: 527-532). These mutations, especially for KRAS, are present at even higher rates in pancreatic cancer and colorectal cancer (KRAS+ in 90% and 50%, respectively, Downward, 2003, and Smalley, 2003). Other studies also reported that NRAS mutations were detected in about 10% to 25% of melanomas (Downard, 2003; Fecher, 2008; Smalley, 2003; Robert, P. J., and Der, C. J., Oncogene, 2007, 26: 3291-3310) and KRAS mutations were detected in about 30% non-small cell lung cancers (NSCLCs) (Downward, 2003; Wistuba, I. I., et al., Semin. Oncol., 2001, 28(2)(suppl 4): 3-13). In addition, RAS mutations (HRAS, KRAS, or NRAS) have been identified in about 55% to 60% of thyroid cancers (Fecher, 2008).
Similarly, BRAF mutations have been identified in about 60% of malignant melanomas, where all mutations appear to be within the kinase domain and a single substitution (T→A, V600E) accounts for about 80% of the mutations (Davies, H., et al., Nature, 2002, 417: 949-954; Kohno, M., and Pouyssegur, J., Ann. Med., 2006, 38: 200-211). Activating BRAF mutations have also been documented in a variety of human cancers other than melanoma, including about 10% in colorectal cancer (CRC), approximately 50% in thyroid cancer (Roberts and Der, 2007), and several percent in NSCLC (Brose, M. S., et al., Cancer Res., 2002, 62: 6997-7000). The high frequency of RAS or BRAF mutations in these cancers makes targeting this pathway an attractive strategy for new anti-cancer agents that rely on patient stratification to identify individuals most likely to benefit from inhibitors that target the MAPK/ERK pathway (Pratilas, C. A., and Solit, D. B., Rev. Recent Clin. Trials, 2007, 2: 121-134).
Scientific and clinical attention has recently focused on the major mutational hotspots in these genes (KRAS codons 12, 13 and 61 and BRAF 600). However, there is increasing evidence that mutations in other locations can also lead to a tumorigenic phenotype (Andre, T., et al., Annals of Oncology, 2012, 00:1-8; DeRook, W., et al., Lancit Oncology, 2010, 11: 6753-6762).
Sanger sequencing, a method of DNA sequencing based on the selective incorporation of chain terminating dideoxnucleotides by DNA polymerase during in vitro DNA replication, considered to be the “gold standard” for detecting genetic mutations, is generally considered not sensitive enough to detect genetic mutations present at low level or low copy number relative to wild type DNA. Commercially available BRAF and KRAS kits, such as, TheraScreen® (CE-IVD) KRAS Mutation kit (DxS Ltd., Manchester, UK) and the FDA approved Cobas® KRAS and BRAF Mutation Test kits (Roche Diagnostics, Indianapolis, Ind.) for testing mutations in codons 12, 13, and 61 of KRAS and the BRAF V600E mutation, respectively, detect mutations only at limited locations. The TheraScreen® KRAS assay is based on real-time PCR Scorpion and ARMS technologies that detects seven frequently encountered mutations in codon 12 and 13 (Angulo, B., et al., J. Mol. Diagn., 2010 May 12(3): 292-299). The Cobas® KRAS Mutation assay, a TaqMelt PCR assay, reports only mutations in general to codon 12/13 or 61, but not specific mutations needed for exploratory data analysis. The Cobas® BRAF Mutation assay only detects the single BRAF V600E mutation. There is currently no commercial NRAS mutation detection assay.